Our Section on Molecular Biophysics measures, characterizes and codifies the forces that govern the organization of all kinds of biological molecules. Our undertaking is strengthened by its strong connection with physical theory. Our long-term goal is to build a practical physics of biological material. We aim to realize this goal through a series of measurements and analyses of the different kinds of forces as revealed in vivo, in vitro and in computation. In particular, we are working with DNA/lipid assemblies for gene therapy; DNA assemblies such as are seen in viral capsids and in vitro; polypeptides and polysaccharides in suspension; and lipid/water liquid-crystals. In all these systems, we simultaneously observe the structure of packing as well as measure intermolecular forces or interaction energies. Van der Waals forces: Weak compared to covalent bonds and to electrical-charge interactions, charge-fluctuation or van der Waals forces are not usually appreciated for the work they do among membranes and macromolecules. The dominant force that coheres membranes and proteins, source of the powerful surface tension at membrane interfaces, van der Waals forces are again the dominant -- perhaps sole -- attraction that creates membrane multilayers or allows membranes to adhere to artificial surfaces. The two past years have been a festival of learning to formulate, to measure, and to modify these neglected interactions. The key has been to begin with the elements of physical theory and to relate the polarizability of materials to the fluctuations of charges within them. From this we have been able to design experiments that show how macromolecular organization responds to deliberate changes in solution properties. We reassessed the Lifshitz theory of van der Waals interactions in stratified media like lipid multilamellar systems and analyzed how the number of lipid bilayers changes the effective interactions between the lamellae within the multilamellar system as well as between a multilamellar system and a wall. These investigations are of primary importance in understanding the behavior of lipid multilayers close to substrates and adsorbing walls. Protein folding: Driven by the conditions set by smaller solutes, proteins "fold" and "unfold." Experimentally, these conditions are stated as intensive variables -- pH and other chemical potentials -- as though small solutes were infinite resources that come at an independently varied price. Computationally, the finite spaces of simulation allow only fixed small numbers of these solutes. By combining the analytic Gibbs adsorption isotherm with the computational Monte Carlo sampling of polymer configurations, we have been able to overcome an inherent limitation of computer simulation. The trick is to compute analytically the free energy changes wrought by solutes on each particular configuration. Then numerical computation is needed only to sample the set of configurations as efficiently as though no bathing solute were present. The result is a more accurate computation procedure five hundred times faster than earlier simulations that had to count all possible positions of the cosolutes in the bathing solution. Solute control of molecular association: To monitor at how small adherent molecules affect molecular association, we measured the changes of binding free energy vs. change in water activity for the specific binding of cyclodextrin with an adamantane derivative. The dependence of the binding constant on osmotic pressure, using different salts and neutral agents, suggests a release of 15-25 water molecules from the interacting surfaces upon association, depending on the type of solute used. The observed dependence of binding free energy and enthalpy with added solute indicates that these osmolytes are interacting primarily enthalpically with these surfaces. A remarkable number of cellular processes are controlled by the osmotic action of small solutes. These include gating of ionic channels and specific versus non-specific DNA?protein interactions regulating gene expression. Osmotic sensing at the molecular level can probe the forces acting between and within macromolecules. By varying the salt or neutral ?osmolyte? concentration in the bathing solution, we control osmotic pressure. We measure the effect of the varied pressure on the association of carbohydrates with membrane protein channels. A single event of b-cyclodextrin (CD) nesting in the lumen of a maltoporin channel is seen as a transient drop in ionic current due to the partial occlusion of the channel pore. The change in equilibrium constant of CD binding to the channel vs. solution osmotic pressure translates into the number of water molecules released in the specific binding. Osmotic pressure differently affects the on- and off-rates of CD-ion channel binding. By changing the species of salt used to exert the osmotic stress, we further probe the properties of the hydration water and the interactions of different salts with both CD and porin. We find that, under equilibrium conditions, the degree to which a particular ion affects the binding is related to the ion?s ranking in the Hofmeister series. In fact, using osmometry we have been able to determine that CD itself is hydrated by waters that are unavailable for the dissolution of salt. Ion-membrane interactions: Because ions vary widely in their effects on biological materials, ion "specificity" beyond simple charge properties is a major issue in biology. One overlooked property of ions is the polarizability, the ability of the charge to shift or fluctuate, a property seen in charge fluctuation forces. Another surprising feature of ions is the tendency to stick to charged bilayers, stick to an extent beyond what is expected from charge-charge attraction. Our comparisons between bilayers of PS and PC lipids with the same chains and the same temperature enable us to focus on the effects of these headgroups on bilayer properties. We find that the charged bilayers repel as expected when interlamellar forces are measured by osmotic stress and x-ray diffraction. Why does this same repulsion not occur between charged groups on the same bilayer? Why are the charged lipids stiffer against bending? The culprit is likely strong ion binding. Indeed we found a few years ago that protons bind so strongly to these same lipids that at 10-4 Molar concentrations (pH 4), the membrane is wrenched from the lamellar form into an inverted hexagonal structure. More intriguing, when these same lipids are used as a scaffold for protein channels, exposure to protons shifts channel gating. Expected from theory and simulations, depletion of ions at fuzzy biomembrane interfaces has long eluded experiments. We have now shown how salt exclusion can be accurately measured by surprisingly simple yet accurate bench-top measurements. Multilamellar aggregates of common phospholipids sink in low salt but float in salt solutions that are much less dense than the lipid itself. By manipulating bath and lipid densities, using heavy water and varied lipid chain length, we have obtained accurate exclusion curves over a wide range of KCl and KBr concentrations. While maintaining a constant width at low salt, the exclusion layer decreases in high salt, following the Debye screening length. Consistent with interfacial accumulation of polarizable ions, bromide salts are less excluded than chloride, with an attraction of ~kT per Br- ion. So far neglected in theoretical descriptions, the competition between salt exclusion and binding is critical to understanding membrane interactions and specific ionic effects.